Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is the most common cause of PH worldwide, affecting approximately 1.5 million patients with heart failure in the United States alone. At present, no specific therapy has been identified mainly due to the fact that major pathways involved in the regulation of PH-HFpEF are still not well understood. We have recently found convincing evidence that PH-HFpEF-regulating sirtuin-3 (SIRT3) is predominantly decreased in the skeletal muscle. In fact, our new preliminary data show that the absence of SIRT3 in skeletal muscle leads to a drastic reduction of the pulmonary vascular tree, accompanied by increased vascular remodeling and higher pulmonary pressures, indicating a critical role of skeletal muscle SIRT3 in pulmonary vascular remodeling and PH-HFpEF. Based on a global mass spectrometry-based comparative secretome analysis, our new data indicate that skeletal muscle SIRT3 deficiency drives the secretion of lysyl oxidase homolog 2 (LOXL2), a matrix enzyme known to promote cross-linking of collagen, and gremlin-1 (Grem1), a bone morphogenetic protein (BMP) antagonist known to induce excessive proliferation. We hypothesize that skeletal muscle SIRT3 deficiency promotes PH-HFpEF by secreting LOXL2 and Grem1, which act as endocrine signaling molecules to induce pulmonary vascular remodeling. We plan to use a number of cutting-edge approaches, which include the use of ex vivo precision-cut lung slices and in vivo skeletal muscle-specific KO mice, to address the outstanding questions. Specific Aims: 1) To determine whether skeletal muscle SIRT3 deficiency in the metabolic syndrome contributes to the development of PH-HFpEF. Using wild-type and skeletal muscle-specific Sirt3 KO mice, with or without high-fat feeding (HFD), which induces a PH-HFpEF phenotype, we will characterize the contribution of skeletal muscle SIRT3 deficiency to the development of PH-HFpEF. Results may reveal a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH- HFpEF. 2, 3) To determine whether LOXL2 and Grem1 are endocrine signaling molecules produced by SIRT3- deficient skeletal muscle cells to induce pulmonary vascular remodeling. Conditioned media from SIRT3- deficient skeletal muscle cells, with or without LOXL2 and Grem1 inhibition, will be applied to ex vivo precision- cut lung slices to test if SIRT3 deficiency with subsequent elevated secretion of LOXL2 and Grem1 promotes vascular remodeling via increased collagen cross-linking and excessive proliferation of pulmonary artery endothelial and smooth muscle cells. The role of skeletal muscle-secreted LOXL2 and Grem1 in pulmonary vascular remodeling and PH-HFpEF will be determined in vivo using skeletal muscle-specific Loxl2 and Grem1 KO mice with HFD. LOXL2 and Grem1 targeting therapies will also be evaluated in our recently developed rat model of PH-HFpEF. We aim to firmly establish the novel paradigm of a skeletal muscle-lung vasculature crosstalk in PH-HFpEF through endocrine signaling. We expect that successful execution of the proposed research plan would provide new mechanistic understanding of PH-HFpEF and identify skeletal muscle SIRT3, along with its downstream LOXL2 and Grem1, as potential molecular targets for the diagnosis and treatment of PH-HFpEF. Furthermore, our studies would provide new insights into how metabolic syndrome, aging, and exercise inactivity affect pulmonary vascular health.